Vitamin D Binding protein (DBP) is a multifunctional and highly polymorphic plasma protein synthesized primarily in the liver (see White, P. et al., Trends Endocrinol. Metabol. 11, 320-327 (2000); Gomme, P. T. et al., Trends Biotechnol. 22, 340-345 (2004)). DBP is expressed as a single polypeptide chain with a molecular mass of approximately 56 kDa and circulates in plasma at 6 to 7 μM (see White, P. et al.; Gomme, P. T. et al.). Due to its extensive polymorphisms DBP initially was named the group-specific component of serum, later shortened to Gc-globulin (see Hirschfeld, J. Acta Pathol. Microbiol. Scand. 47, 160 (1959)). DBP is a member of the albumin (ALB), alpha-fetoprotein (AFP), and alpha-albumin/afamin (AFM) gene family and hence has the characteristic multiple disulfide-bonded, triple domain modular structure (see White, P. et al.). Besides functioning as a circulating vitamin D transport protein, it has been demonstrated that plasma DBP effectively scavenges G-actin released at sites of necrotic cell death and prevents polymerization of actin in the circulation (see White, P. et al.; Gomme, P. T. et al.). Distinct binding regions within the 458 amino acid sequence of DBP have been identified: a vitamin D sterol binding segment in the N-terminal domain (amino acids 35 to 49) and a G-actin binding region in the C-terminal domain (amino acids 373 to 403) (see Haddad, J. G. et al., Biochemistry 31, 7174-7181 (1992); Swamy, N. et al., Biochemistry 36, 7432-7436 (1997)). More recent work on the crystal structure of DBP (bound to either vitamin D3 or actin) has confirmed the vitamin D sterol binding site, but has demonstrated that actin interacts with distinct amino acid sequences in all three DBP domains (see Verboven, C. et al., Nat. Struct. Biol. 9, 131-136 (2002); Swamy, N. et al., Arch. Biochem. Biophys. 402, 14-23 (2002); Head, J. F. et al., Biochemistry 41, 9015-9020 (2002); Otterbein, L. R. et al., Proc. Natl. Acad. Sci. USA 99, 8003-8008 (2002)).
Complement C5a is a 74-amino acid peptide generated by limited proteolytic cleavage of C5 during complement activation (see Kohl, J. Mol. Immunol. 38, 175-187 (2001)). C5a is a very potent chemotactic factor for all leukocytes as well as several other cell types and the peptide has several other pro-inflammatory functions as well ( see Kohl, J.). C5a exerts these activities by binding to its high affinity receptor (C5aR or CD88) on the plasma membrane of target cells (see Gerard, N. P. et al. Nature. 349, 614-617 (1991)).
Several groups have demonstrated that purified DBP can significantly enhance the neutrophil chemotactic activity (i.e., cochemotatic activity) of C5a, and its stable breakdown product C5a des Arg (see Kew, R. R. et al., J. Clin. Invest. 82, 364-369 (1988); Perez, H. D. et al., J. Clin. Invest. 82, 360-363 (1988); Petrini, M. et al., J. Endocrinol. Invest. 14, 405-408 (1991); Metcalf, J. P. et al., Am. Rev. Respir. Dis. 143, 844-849 (1991); Binder, R. et al., Mol. Immunol. 36, 885-892 (1999); Zwahlen, R. D. et al., Inflammation 14, 109-123 (1990)). In addition to neutrophils, DBP can also augment the C5a chemotactic activity for monocytes and fibroblasts (see Piquette, C. A. et al., J. Leukoc. Biol. 55, 349-354 (1994); Senior, R. M. et al., J. Immunol. 141, 3570-3574 (1988)). The chemotactic enhancing properties of DBP appear to be restricted to C5a/C5a des Arg since this protein cannot enhance the chemotactic activity of formylated peptides, IL-8, leukotriene B4 or platelet activating factor (see Kew, R. R. et al.; Perez, H. D. et al.; Binder, R. et al.; Piquette, C. A. et al.).
Surface plasmon resonance (SPR) is a powerful state-of-the-art technology that enables investigators to measure the interaction between molecules in real time. The leader in this technology is Biacore AB (Uppsala, Sweden) and the SPR applications are very often referred to using the company name. Using Biacore, the molecule of interest (ligand) is immobilized to a sensor chip. The sensor chip is placed in a flow cell and a second soluble molecule (analyte) is allowed to interact with the immobilized ligand. The strength (association/dissociation constant) and speed (on/off rate) of the molecule interaction can be calculated in real time. Theoretically, Biacore can be used to measure cell binding to an immobilized ligand, however, very little information is available concerning this type of application.
Undifferentiated U937 cells stably transfected with the C5a receptor (U937-C5aR cells) are a good model system to study C5a-mediated cell movement and the chemotactic enhancing effect of DBP. However, the effect of DBP on C5a-mediated increase in intracellular calcium concentrations has not been determined.